DNA damage response (DDR) is a complex signal transduction pathway, which is extremely important to maintain genomic stability and prevent carcinogenesis by counteracting the deleterious effects of DNA damage. In response to replication stress or UV irradiation, the primary event of DDR is to activate kinases ATR and Chk1, which in turn trigger multiple physiological processes, including DNA synthesis inhibition, DNA repair, and cell cycle delay, transcription, etc. ATR plays a key role in Chk1 activation and DNA synthesis inhibition in response to DNA damage. However, how ATR coordinates with various DDR components to activate Chk1 and how ATR targets replication apparatus to inhibit DNA synthesis remain largely unknown in mammalian cells. We have demonstrated that human And-1 acts as a key component of replisome at replication forks for DNA replication. We now have solid preliminary data indicating that And-1 is also involved in the regulation of checkpoint and DNA synthesis in response to DNA damage. Thus, elucidating the role of And-1 in DDR will fill in critical knowledge gaps of DDR signaling. Building upon our recently published work and our extensive new data, in this proposal we described a series of innovative, hypothesis-driven studies to elucidate how human And-1 regulates checkpoint activation and DNA synthesis in response to UV irradiation and replication stress. First, we will determine the mechanism of how And-1 is recruited to DNA damage sites using both biochemical and structural analyses. Second, we will determine how And-1 acts as a unique replisome component to regulate checkpoint activation by impacting interplay among multiple key DDR components including ATR, Rad17, Claspin, and Timeless-Tipin at stalled replication forks using molecular and structural analyses. Finally, we will determine mechanism by which And-1 governs DNA synthesis in response to DNA damage by carrying out an innovative approach that combines multiple cutting edge technologies including single-molecule analyses, iPOND, and mass spectrometry. Given that targeting the circuitry of DNA damage response has been a key focus for the development of anti-cancer drugs, proposed work will not only advance the field by identifying new components and acquiring in-depth mechanistic understanding of DNA damage response, but also provide us with new strategies for the development of highly specific anti-cancer therapies targeting And-1 or And-1-dependent processes.